PerspectiveCell Biology

Caspase Inhibitors Promote Alternative Cell Death Pathways

See allHide authors and affiliations

Science's STKE  24 Oct 2006:
Vol. 2006, Issue 358, pp. pe44
DOI: 10.1126/stke.3582006pe44

Abstract

The use of caspase inhibitors has revealed the existence of alternative backup cell death programs for apoptosis. The broad-spectrum caspase inhibitor zVAD-fmk modulates the three major types of cell death. Addition of zVAD-fmk blocks apoptotic cell death, sensitizes cells to necrotic cell death, and induces autophagic cell death. Several studies have shown a crucial role for the kinase RIP1 and the adenosine nucleotide translocator (ANT)–cyclophilin D (CypD) complex in necrotic cell death. The underlying mechanism of zVAD-fmk–mediated sensitization to necrotic cell death involves the inhibition of caspase-8–mediated proteolysis of RIP1 and disturbance of the ANT-CypD interaction. RIP1 is also involved in autophagic cell death. Caspase inhibitors and knockdown studies have revealed negative roles for catalase and caspase-8 in autophagic cell death. The positive role of RIP1 and the negative role of caspase-8 in both necrotic and autophagic cell death suggest that the pathways of these two types of cell death are interconnected. Necrotic cell death represents a rapid cellular response involving mitochondrial reactive oxygen species (ROS) production, decreased adenosine triphosphate concentration, and other cellular insults, whereas autophagic cell death first starts as a survival attempt by cleaning up ROS-damaged mitochondria. However, when this process occurs in excess, autophagy itself becomes cytotoxic and eventually leads to autophagic cell death. A better understanding of the molecular mechanisms of these alternative cell death pathways may provide therapeutic tools to combat cell death associated with neurodegenerative diseases, ischemia-reperfusion pathologies, and infectious diseases, and may also facilitate the development of alternative cytotoxic strategies in cancer treatment.

Cell death is a crucial process in the development and homeostasis of multicellular organisms. Moreover, deregulation of this process contributes to major pathologies, including cancer, autoimmune diseases, neurodegenerative diseases, and ischemia-reperfusion damage. Three major morphologies of cell death have been described: apoptotic, necrotic, and autophagic cell death. Hallmarks of apoptotic cell death include activation of caspases, DNA fragmentation, and membrane blebbing (1). Necrosis is associated with swelling of the cell and its organelles, leading to loss of plasma membrane integrity (2). Autophagic cell death is recognized by the formation of autophagosomes, double-membrane autophagic vacuoles that eventually fuse with lysosomes to form autolysosomes (3).

Death Domain Receptor–Mediated Cell Death

Death receptors belonging to the tumor necrosis factor (TNF) receptor superfamily mediate many different cellular processes, ranging from cell survival to inflammatory and cell death responses (4). TNF, a pleiotropic cytokine produced primarily by macrophages, stimulates TNF receptor 1 (TNFR1)–mediated activation of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) in most cell types, but in cancer cell lines, mostly in the presence of translation inhibitors, it causes apoptotic cell death (5, 6). In the case of TNFR1, these differential activities are mediated by the formation of two different receptor complexes. At the plasma membrane, formation of complex I—which consists of TNFR1, TRAF2 (TNFR-associated factor 2), and RIP1 (receptor interacting protein 1)—leads to rapid activation of NF-κB and MAPKs, such as p38 MAPK, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK). After receptor endocytosis, a second complex is formed in which the adaptor protein TRADD recruits FADD, another adaptor protein, and procaspase-8 or procaspase-10, thereby mediating apoptosis (79). It is unclear what regulates the switch between the formation of the proinflammatory and the proapoptotic complexes.

In some cell types, TNF elicits necrotic cell death either spontaneously or when caspases are blocked by addition of synthetic caspase inhibitors or by overexpression of CrmA, a cowpox caspase-1 and caspase-8–specific inhibitor (10). Moreover, the absence of FADD or caspase-8 in Jurkat cells apparently also favors TNF-mediated necrosis (11). These results indicate that necrotic cell death may function as a backup cell death pathway when caspases are blocked or when the caspase-dependent pathways cannot be properly activated. Additionally, the presence of caspase inhibitors sensitizes cells to necrotic cell death (10) in response to TNF, which suggests a negative interplay between caspase activation and necrotic cell death as well.

Role of RIP1 Kinase in Necrotic Cell Death

Several lines of evidence suggest that the death domain–containing serine-threonine kinase RIP1 is important for necrotic cell death. Studies in RIP1−/− Jurkat cells demonstrated that necrosis induced by triggering of TNFR1, Fas, or TRAIL-R depends on the presence of RIP1 (9, 12, 13). Moreover, RNA interference (RNAi)–mediated knockdown of RIP1 in L929 cells retards necrotic cell death induced by TNF in the presence of zVAD-fmk or FasL in the presence of zVAD-fmk (14). zVAD-fmk (benzoyl-Val-Ala-Asp-fluoromethyl ketone) is a broad-spectrum caspase inhibitor that prevents apoptotic cell death. The involvement of RIP1 in death receptor–induced necrosis was confirmed in studies of heat shock protein (Hsp) 90, a cytosolic chaperone for many kinases, including RIP1 (15). Inhibition of Hsp90 by geldanamycin or radicicol, a structurally unrelated inhibitor, leads to a factor of 10 down-regulation of RIP1 expression levels and inhibits necrosis triggered by the receptors Fas and TNFR1 (12, 16). Moreover, caspase-8–mediated cleavage of RIP1 during apoptosis triggered by TNFR1, Fas, or TRAIL-R suppresses not only necrotic signaling but also antiapoptotic pathways by abrogation of RIP1-induced NF-κB activation (17, 18).

Role of PARP-1 in Necrotic Cell Death

In addition to death receptor–induced, caspase-independent cell death, stimulation of Toll-like receptors 3 or 4 (TLR3 or TLR4) in the presence of caspase inhibitors also stimulates caspase-independent, RIP1-mediated death (11, 19). Moreover, some pathophysiological processes, such as injury caused by ischemia-reperfusion, inflammation, reactive oxygen species (ROS), and glutamate excitotoxicity, are accompanied by poly(ADP-ribose) polymerase-1 (PARP-1)–mediated caspase-independent cell death. This PARP-1–mediated death is due to nicotinamide adenine dinucleotide (NAD) consumption and reduced adenosine triphosphate (ATP) generation (2022). Thompson and colleagues demonstrated that, relative to cells that catabolize nonglucose substrates to maintain oxidative phosphorylation, cells that use aerobic glycolysis are more susceptible to PARP-1–mediated cell death elicited by DNA alkylating agents (23). This may explain the sensitivity of many tumor cells to DNA-damaging agents, as cancer cells maintain their ATP production mostly through aerobic glycolysis. Interestingly, PARP-1–mediated cell death induced by H2O2 (24) also depends on a TRAF2-RIP1-JNK signaling cascade (25); this finding suggests that diverse cellular stress conditions, glucose metabolism, and ATP sentinels impinge on RIP1 activation. Together, these data demonstrate that necrotic cell death is also an active cell death process that is governed by signal transduction and metabolic pathways.

Switches Between Necrotic and Autophagic Cell Death

Recently, some studies reported on the induction of RIP1-dependent autophagic cell death instead of necrosis after exposure to zVAD-fmk or specific RNAi-mediated caspase-8 knockdown in L929 cells (26, 27), suggesting that the absence of caspase activity can also favor the induction of autophagic cell death. However, the induction of autophagic cell death in L929 cells is much slower than the induction of death receptor–induced necrotic cell death in these cells. Additionally, macrophages can die by necrosis or autophagic death after treatment with lipopolysaccharide (LPS) in the presence of caspase inhibitors (19, 28). Thus, whether necrosis or autophagy ensues when apoptosis is inhibited may depend on cells and circumstances. Because of the lack of specific positive markers of necrotic cell death and the fact that autophagy may always occur in conditions of cellular stress, these types of death may be frequently entangled. White and co-workers demonstrated that inhibition of autophagy in apoptosis-refractory solid tumors promotes necrotic cell death and inflammation, which may stimulate tumor cell proliferation; this result indicates that autophagy can negatively regulate necrotic cell death as a tumor survival mechanism (29). In tumors, as is the case for physical injury, necrosis is associated with inflammation. Inflammation is part of wound healing, where cellular infiltration and chemokine and cytokine production function to stimulate proliferation, tissue remodeling, and angiogenesis. The inflammatory response to stress-mediated, oncogene-activated necrosis in tumors, by analogy to a wound-healing response, may stimulate angiogenesis and tumor cell proliferation (29).

Molecular Mechanisms of zVAD-fmk– and RIP1-Mediated Necrotic and Autophagic Cell Death

The results discussed above suggest that the kinase RIP1 and caspase inhibition by zVAD-fmk play a crucial role in both alternative cell death pathways, necrosis and autophagic death. When the mechanism of action of a compound such as the caspase inhibitor zVAD-fmk is evaluated, one should take into account the concentration used and confirm the observed effect by using a natural inhibitor (for example, CrmA) or by eliminating the target caspase, in this case caspase-8. With the use of these approaches, a paradigm is emerging in which caspase-8 can negatively regulate necrotic signaling at least partly by specific cleavage of RIP1 (17) (Fig. 1). The same holds true for autophagic cell death; the presence of zVAD-fmk or absence of caspase-8 is sensitizing, whereas the absence of RIP1 prevents autophagic cell death (26).

Fig. 1.

The broad-spectrum caspase inhibitor zVAD-fmk modulates the three major types of cell death in different ways. zVAD-fmk blocks apoptotic cell death while it sensitizes cells to necrotic cell death. zVAD-fmk is also reported to induce autophagic cell death. Autophagy and necrotic cell death are interconnected and may partially consist of common underlying molecular pathways involving RIP1 and negative regulation by caspase-8. In addition, a positive role for cyclophilin D (CypD) and a negative role for catalase in caspase-independent cell death cascades have been demonstrated (see text for more details).

Another target of zVAD-fmk has been identified: the adenosine nucleotide translocator (ANT), an inner mitochondrial membrane transport protein involved in the export of ATP to the cytosol and the import of adenosine diphosphate (ADP) into the mitochondria. Temkin and colleagues (30) reported that zVAD-fmk at a concentration of 20 μM targets, as it does with caspases, a cysteinyl residue in ANT (Cys56), preventing the interaction with cyclophilin D (CypD), which is required for proper functioning of ANT (Fig. 1). Interestingly, addition of TNF alone is able to prevent the interaction between ANT and CypD in a RIP1-dependent manner. Moreover, in TNF-stimulated cells, RIP1 translocates to the mitochondria, which suggests a possible role of this death domain kinase in the displacement of CypD from ANT. The direct consequence of improper functioning of ANT is enhanced ROS production and diminished ATP production. This crucial role of CypD in necrotic cell death has been confirmed in cells from CypD-deficient mice. These mice died normally in response to various apoptotic stimuli, but showed resistance to necrotic cell death induced by ROS or Ca2+ overload. In addition, CypD-deficient mice were resistant to cardiac injury due to ischemia and reperfusion. These results show that CypD regulates at least some forms of necrotic death, but not apoptotic death (3133).

As mentioned above, activation of PARP-1 catalyzes the hydrolysis of NAD+ into nicotinamide and poly(ADP-ribose), causing depletion of NAD+ (34). This decrease in oxidized equivalents eventually results in reduced oxidative phosphorylation, which is similar to the effect of nonfunctional ANT. Nicotera et al. (35) documented a role for decreased concentration of ATP in the induction of necrotic cell death. However, it remains unclear whether all necrotic cell death is accompanied by a drop in ATP concentration. TNF-induced necrosis of L929 cells in the absence of zVAD-fmk proceeds with normal concentrations of ATP (36). Furthermore, translation persists during necrosis of Jurkat and L929 cells, which also argues against a massive drop in ATP concentrations during necrotic cell death (37).

The induction of autophagic cell death by addition of zVAD-fmk or absence of caspase-8 (26) was also associated with enhanced mitochondrial ROS production. A possible mechanism has been proposed by Lenardo and colleagues; they demonstrated that caspase inhibition directly induces catalase degradation and mitochondrial ROS accumulation, suggesting a protective role of caspase-8 against oxidative stress in mitochondria (27) (Fig. 1). This survival role of caspase-8 is revealed by the fact that caspase-8–deficient mice embryos die at embryonic day 11.5 (E11.5) as a result of neural and heart defects, which suggests a protective role of caspase-8 in embryogenesis (38, 39).

Although the proposed molecular mechanisms are very different—inhibition of caspase-8, competition for CypD binding to ANT, or induction of catalase degradation—the eventual effect of zVAD-fmk administration is enhanced ROS production, leading to either necrosis or autophagic cell death. Although the mechanism of cell death seems to be completely different, ROS production, energy metabolism, and active caspases may determine whether the cell will go directly into necrosis or will first try to restore the damage by autophagy. If cellular stress continues, the autophagy may eventually result in cell death.

Conclusions

The use of caspase inhibitors has revealed the existence of alternative cell death pathways, which may function as backup cell death programs for apoptosis. Apparently, the caspase inhibitor zVAD-fmk modulates the three major types of cell death in different ways. Addition of zVAD-fmk blocks apoptotic cell death while sensitizing cells to necrotic cell death. Autophagic cell death may also be triggered by zVAD-fmk. Several studies demonstrated the important role of RIP1 in the initiation of caspase-independent death. In view of the intriguing relocation of RIP1 to the mitochondria (30), the identification of RIP1 substrates will be especially enlightening. Interestingly, the use of caspase inhibitors has revealed a positive role for CypD and negative roles for catalase and caspase-8 in caspase-independent cell death cascades. Necrotic and autophagic cell death seem to be interconnected and may partially consist of common underlying molecular pathways involving RIP1 and negative regulation by caspase-8. Necrotic cell death may represent a rapid cellular response involving mitochondrial ROS production, decreased ATP concentration, and other cellular insults, whereas autophagic cell death may first start as a survival attempt, blocking necrosis (29) and cleaning up oxidatively damaged mitochondria (40) by a process now called mitoptosis (41). However, when this process occurs in excess, autophagy itself becomes cytotoxic and eventually leads to autophagic cell death. A better understanding of the molecular mechanisms of these alternative cell death pathways may provide therapeutic tools to combat cell death associated with neurodegenerative diseases, ischemia-reperfusion pathologies, and infectious diseases, and to develop alternative cytotoxic strategies in cancer treatment.

References

  1. 1.
  2. 2.
  3. 3.
  4. 4.
  5. 5.
  6. 6.
  7. 7.
  8. 8.
  9. 9.
  10. 10.
  11. 11.
  12. 12.
  13. 13.
  14. 14.
  15. 15.
  16. 16.
  17. 17.
  18. 18.
  19. 19.
  20. 20.
  21. 21.
  22. 22.
  23. 23.
  24. 24.
  25. 25.
  26. 26.
  27. 27.
  28. 28.
  29. 29.
  30. 30.
  31. 31.
  32. 32.
  33. 33.
  34. 34.
  35. 35.
  36. 36.
  37. 37.
  38. 38.
  39. 39.
  40. 40.
  41. 41.
  42. 42.
View Abstract

Navigate This Article